PROJECT SUMMARY/ABSTRACT For more than 100 years, diabetes has been considered an immune-suppressed condition. In contrast, we hypothesize that the opposite is true: worse outcomes of Gram-negative infection in diabetics are due to diabetes-driven enhanced immunity because: 1) Toll-like receptor 4 (TLR4) is anti-protective during infection caused by Gram negative bacteria; 2) TLR4 inflammatory output in response to LPS is exacerbated in diabetics; 3) we recently found that blocking the binding of Advanced Glycation Endproducts (AGE) to their receptor (RAGE) ameliorated hyper-susceptibility of diabetic mice to Gram-negative infection; and 4) both TLR4 and RAGE can signal via MyD88, suggesting a mechanism for RAGE/TLR4 synergy. Our central premise?diabetes enhances rather than suppresses the innate response to infection?has novel translational impact. Specifically, immunomodulation to improve outcomes of infection in diabetic hosts should seek to reverse a 100 year-old maxim: suppress the innate response rather than enhance it. We seek to: 1) determine the RAGE/TLR4 signaling relationships during I.V. and lung infections caused by Gram-negative bacteria; and 2) develop translational solutions using promising compounds to normalize immune responses, reducing morbidity and mortality. Our Aims are to: Specific Aim 1: Define the role of MyD88- vs. TRIF-signaling during I.V. and lung infection in diabetic mice. HYPOTHESIS: MyD88 is the signaling pathway mediating susceptibility to Gram-negative infection in diabetic mice, since it can independently signal for TLR4 and RAGE, creating potential for synergistic inflamm- ation. Methods: MyD88 and TRIF are the primary pathways responsible for TLR4 signaling. We found that TLR4 disruption markedly but incompletely protected diabetic mice from A. baumannii infection, possibly because of residual signaling via MyD88 by RAGE. To define the pathway driving outcome, will compare survival, sepsis biomarkers, and bacterial burden during I.V. and lung infection in wild type (pos. control) vs. TLR4-KO (neg. control), MyD88-KO, and TRIF-KO mice that are diabetic or treated with a RAGE-agonist. Specific Aim 2: Determine the potential for a combination of inhibitors presently in pharmaceutical development against TLR4 and RAGE to alter outcomes during Gram-negative bacterial infection in diabetic hosts. HYPOTHESIS: Simultaneous inhibition of TLR4 and RAGE will provide superior outcomes from infection. Methods: We will compare survival, sepsis biomarkers, and bacterial burden in non-diabetic (neg. control) vs. diabetic mice infected I.V. or through the lungs, and treated with placebo (pos. control) vs. 1) a TLR4 antagonist; 2) a RAGE antagonist; or 3) combination of both. To ensure generalizability, mice will be infected with three antibiotic-resistant bacteria: A. baumannii, E. coli, and Klebsiella pneumoniae. IMPACT: These results will enable translation of immune therapies for these deadly infections, and will enable an R01 to define downstream RAGE signaling and metabolic mechanisms driving inflammation in diabetes.